Anxiolytic-like effect of brominated compounds from the marine sponge Aplysina fulva on adult zebrafish (Danio rerio): Involvement of the GABAergic system.

Benzodiazepines are commonly used to treat disorders of the central nervous system, including anxiety. However, due to their adverse effects, there is a continuing interest in discovering new safe and effective drugs. Marine natural products have emerged as a prolific source of bioactive nitrogenated compounds. Aiming to discover new biologically active natural compounds, the marine sponge Aplysina fulva, a nitrogen-bearing heterocyst producer, was investigated. The main isolated compounds (4, 6, and 9) were evaluated on adult zebrafish (Danio rerio). A group of fishes (n = 6) was preliminarily subjected to acute toxicity, and open field tests using 0.1, 0.5, and 1.0 mg/mL (v. o.) of those compounds was performed. The anxiolytic effect was further investigated in the light/dark assay based on the locomotor response at zebrafish. Interactions through the GABAergic system were investigated using flumazenil, a silent modulator of GABA receptors. To improve the results, a study of molecular docking using the GABAA receptor also was performed. Based on the results, the bromotyrosine derivative compounds 4, 6, and 9 exhibited anxiolytic-like effects mediated by the GABAergic system.

Surface active benzodiazepine-bromo-alkyl conjugate for potential GABAA-receptor purification.

A conjugable analogue of the benzodiazepine 5-(2-hydroxyphenyl)-7-nitro-benzo[e][1,4]diazepin-2(3H)-one containing a bromide C(12)-aliphatic chain (BDC) at nitrogen N1 was synthesized. One-pot preparation of this benzodiazepine derivative was achieved using microwave irradiation giving 49% yield of the desired product. BDC inhibited FNZ binding to GABA(A)-R with an inhibition binding constant K(i) = 0.89 µM and expanded a model membrane packed up to 35 mN m(-1) when penetrating in it from the aqueous phase. BDC exhibited surface activity, with a collapse pressure π = 9.8 mN m(-1) and minimal molecular area A(min) = 52 Å(2)/molecule at the closest molecular packing, resulted fully and non-ideally mixed with a phospholipid in a monolayer up to a molar fraction x≅ 0.1. A geometrical-thermodynamic analysis along the π-A phase diagram predicted that at low x(BDC) (<0.1) and at all π, including the equilibrium surface pressures of bilayers, dpPC-BDC mixtures dispersed in water were compatible with the formation of planar-like structures. These findings suggest that, in a potential surface grafted BDC, this compound could be stabilize though London-type interactions within a phospholipidic coating layer and/or through halogen bonding with an electron-donor surface via its terminal bromine atom while GABA(A)-R might be recognized through the CNZ moiety.

Anxiolytic-like effect of Carvacrol (5-isopropyl-2-methylphenol) in mice: involvement with GABAergic transmission.

Carvacrol (5-isopropyl-2-methylphenol) is a monoterpenic phenol present in the essencial oil of many plants. It is the major component of the essential oil fraction of oregano and thyme. This work presents the behavioral effects of carvacrol in animal models of elevated plus maze (EPM), open field, Rotarod and barbiturate-induced sleeping time tests in mice. Carvacrol (CVC) was administered orally, in male mice, at single doses of 12.5; 25 and 50 mg/kg while diazepam 1 or 2 mg/kg was used as standard drug and flumazenil (2.5 mg/kg) was used to elucidate the possible anxiolytic mechanism of CVC on the plus maze test. The results showed that CVC, at three doses, had no effect on the spontaneous motor activity in the Rotarod test nor in the number of squares crossed in the open-field test. However, CVC decreased the number of groomings in the open-field test. In the plus maze test, CVC, at three doses significantly increased all the observed parameters in the EPM test and flumazenil was able to reverse the effects of diazepam and CVC. Therefore, CVC did not alter the sleep latency and sleeping time in the barbiturate-induced sleeping time test. These results show that CVC presents anxiolytic effects in the plus maze test which are not influenced by the locomotor activity in the open-field test.

Isolation and identification of 6-methylapigenin, a competitive ligand for the brain GABA(A) receptors, from Valeriana wallichii.

Using the guidance by a competitive assay for the benzodiazepine binding site in the GABA(A) receptor, active compounds were isolated from the rhizomes and roots of Valeriana wallichii DC. The UV, NMR and mass spectral data permitted the identification of 6-methylapigenin. This flavonoid has a Ki = 495 nM for the BDZ-bs and a GABA ratio of 1.6-2.0, which suggests possible agonistic properties. The calculated percentage of 6-methylapigenin in the crude drug is in the range: 0.013% to 0.0013%.

Synthesis and GABA(A) receptor activity of 6-oxa-analogs of neurosteroids.

The 6-oxasteroids 3alpha-hydroxy-6-oxa-5alpha-pregnan-20-one (3) and 3alpha-hydroxy-6-oxa-5beta-pregnan-20-one (4) were obtained from pregnenolone acetate via the corresponding (5alpha or 5beta) 3beta, 20beta-diacetoxy-6-oxa-pregnane. Both steroids showed ca. 100-fold reduced potency for modulating [(3)H]flunitrazepam, [(3)H]muscimol or [(35)S]TBPS binding to the GABA(A) receptor when compared to their natural carbon analogs 3alpha-hydroxy-5alpha-pregnan-20-one (1) and 3alpha-hydroxy-5beta-pregnan-20-one (2).

Partitioning of flunitrazepam into model membranes studied by temperature controlled gel filtration chromatography.

The use of gel filtration chromatography with Sephadex as a separation medium was used in order to study flunitrazepam (FNTZ) partitioning into artificial model membranes consisting of dipalmitoyl-phosphatidylcholine (dpPC) vesicles, under controlled temperature conditions. In this system two phenomena are taking place simultaneously: the ligand-liposome interaction and the lipid self-aggregation to form the liposome. The liposome-FNTZ interaction was evidenced by the non-enantiography of the first derivative of FNTZ elution peak in frontal chromatography through Sephadex G-75. On the other hand, the presence of FNTZ reduced liposomes mean size and increased their size dispersion as evidenced by molecular filtration through Sephadex G-200. The dpPC-buffer FNTZ partition coefficient determined in zonal chromatography through Sephadex G-10 increased about 33% when the temperature rose above the temperature of dpPC transition from the liquid crystalline to the fluid phase. Gel filtration chromatography seems a suitable technique to study lipid liposome-FNTZ interactions at a qualitative level. In addition, this technique has the advantage over other methods of giving the possibility of observing the mutual effects exerted between the drug and the self-aggregating structure.